Identification and Comparative Expression Profiles of Candidate Olfactory Receptors in the Transcriptomes of the Important Egg Parasitoid Wasp Anastatus japonicus Ashmead (Hymenoptera: Eupelmidae)

Anastatus japonicus Ashmead is an egg parasitoid wasp important for the biological control of fruit crop pests. The olfaction of parasitoids is crucial to searching for host pests in fruit crops. In this study, we sequenced and analyzed the antennal and abdominal transcriptomes of A. japonicus to better understand the olfactory mechanisms in this species. A total of 201 putative olfactory receptor genes were identified, including 184 odorant receptors (ORs) and 17 ionotropic receptors (IRs). Then, we assayed the tissue-specific and sex-biased expression profiles of those genes based on the transcriptional levels. In total, 165 ORs and 15 IRs had upregulated expression in the antennae. The expression levels of 133 ORs, including odorant receptor co-receptor (AjapORco), and 10 IRs, including AjapIR8a, were significantly different between the female and male antennae. Our results provide valuable information for further studies on the molecular mechanisms of the olfactory system in A. japonicus.


Introduction
Anastatus japonicus Ashmead (Hymenoptera: Eupelmidae), an important egg parasitoid for biological control, has a wide range of hosts, with over 15 host species in two families of Hemiptera (Alydidae and Pentatomidae) and 5 families of Lepidoptera (Lasiocampidae, Lymantriidae, Notodontidae, Papilionidae, and Saturniidae) [1]. Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) feeds on many fruit crops and causes serious economic losses worldwide [2][3][4]. Releases of A. japonicus against H. halys have been used in an organic kiwifruit orchard with satisfactory effects [5]. Similarly, in recent years, Caligula japonica (Moore) (Lepidoptera: Saturniidae) is a significant pest of walnut, chestnut, plum, apple, pear, and persimmon fruit trees, and its egg parasitoids also include A. japonicus [6]. Anastatus japonicus reared on Antheraea pernyi (Guérin-Méneville) (Lepidoptera: Saturniidae) eggs had a high biological control potential against Caligula japonica [7,8]. Therefore, A. japonicus has become an important biological control factor for fruit crop pests; more importantly, A. japonicus can be raised on A. pernyi eggs on a large scale indoors [8,9]. The ability of parasitoid wasps to search for hosts in complex chemical surroundings greatly affects their success in suppressing pest populations, thus affecting the economic value of fruit crops [10]. Therefore, the ability of A. japonicus to search for crop pests is the key to the efficiency of pest control in the field.
Parasitoid wasps are a major component of Hymenoptera and have considerable ecological effects on the populations of many other insects [11][12][13]. They use various physical and environmental cues to search for hosts in a multistage process, including host under conditions of 25 ± 1 • C, 70 ± 5% RH, and a 14:10 h L:D photoperiod. As previously studied, A. pernyi eggs were used as hosts [2].
Tissues from 2-4-day old adults were dissected under a microscope and placed in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) on ice. Tissues were then stored at −80 • C until use. Antennae were obtained from 1000 adults (500 females and 500 males), and 50 abdomens were obtained from females. Three biological replicates for each tissue were conducted.

cDNA Library Construction and Sequencing
Total RNA from each of the nine samples was extracted using the TRIzol (Invitrogen, Carlsbad, CA, USA) method. Then, we used a 2100 Bioanalyser (Agilent Technologies, Inc., Santa Clara CA, USA) and an ND-2000 (NanoDrop Thermo Scientific, Wilmington, DE, USA) to determine the integrity and purity of the total RNA quality and quantity, respectively. Four micrograms of RNA from each sample were used for cDNA library construction. The nine libraries were sequenced in a single lane on an Illumina NovaSeq 6000 sequencer (Illumina, San Diego, CA, USA) for 2 × 150 bp paired-end reads.

Transcriptome Assembly and Functional Annotation
The raw reads were obtained through high-throughput sequencing and then quality controlled using SeqPrep (https://github.com/jstjohn/SeqPrep, accessed on 2 February 2023) and Sickle (https://github.com/najoshi/sickle, accessed on 2 February 2023) with default parameters. Then, clean data from all the samples were used to for de novo assembly with Trinity v2.4.0 [42]. The raw data were entered into the NCBI Sequence Read Archive with BioProject ID number PRJNA931446. In order to obtain comprehensive gene function information, we performed gene function annotations using BLAST with an e-value < 1 ×10 −5 in some major databases, including the non-redundant protein sequence database (NR), Swiss-Prot, Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), eggNOG, and Pfam [43,44].

Identification of Olfactory Receptor Genes
To identify candidate olfactory receptor genes in A. japonicus, the sequences whose best-hit annotations were ORs or IRs in at least one database were retained as candidate unigenes encoding putative ORs or IRs. Then, the total identified putative ORs or IRs were checked against the NCBI Nr database via BLASTx searches (e-value < 1 × 10 −5 ) manually. The open reading frames (ORFs) and the transmembrane domains (TMDs) of the candidate olfactory receptor genes were analyzed using ExPASy service (http://web.expasy. org/translate/, accessed on 2 February 2023) and Tmhmm 2.0 (http://www.cbs.dtu.dk/ services/TMHMM//, accessed on 2 February 2023) with the default parameters, respectively.

Expression Abundance Analysis of Olfactory Receptor Genes
For the differentially expressed gene (DEG) analysis, the Bowtie alignment method and RSEM were used to align the reads on the transcriptome and to calculate the raw read numbers and TPM (transcripts per kilobase million) expression value [50]. The DEG analysis of the two different samples, i.e., the female abdomen and antennae, was performed using the DESeq2 v1.24 package [51]. The |log 2 (FoldChange)| > 1 and adjusted p value < 0.01 were identified as significant for the DEG. The expression of the OR and IR genes was revealed by a heatmap using the TBtools v1.082 software package and a volcano plot using the GraphPad Prism 9 software package.

Overview of the Anastatus japonicus Transcriptome
The transcriptomes of female and male antennae and female abdomens of A. japonicus were sequenced using the Illumina NovaSeq 6000 platform. In total, 424,066,006 raw reads were obtained from nine cDNA libraries. After filtering the raw data, 416,603,736 clean reads were generated, with Q20 accounting for more than 95.9% ( Table 1). The de novo assembly produced 144,436 transcripts and 132,646 unigenes, with the N50 value of 1544 and 905 bp, respectively. In addition, a final transcript dataset with 17,474 coding genes was produced ( Table 2). Among all the coding genes, 16,661 were successfully annotated, accounting for 95.35% of the total. The largest proportion of annotation in a single database was obtained for NR (93.00%), followed by PFAM (89.44%) and SwissProt (66.87%) ( Table S1).

Identification of Putative Odorant Receptors
We identified 184 putative OR genes in A. japonicus. The sequence analysis revealed that 92 of 184 sequences were full-length putative OR genes with ORFs with five to eight predicted TMDs and an average length of 1163 bp. The OR co-receptor, named AjapOrco, was also found. Other putative ORs (AjapOR1-AjapOR183) were given names followed by a numeral in descending order of the length of their coding regions. Sequence information for the putative ORs in A. japonicus is listed in Table S2 and File S1.
To further assess the relationships between the A. japonicus ORs and the known Hymenoptera ORs, we carried out an ML tree analysis using the putative AjapORs and ORs of T. pretiosum, N. vitripennis, and A. mellifera ( Figure 1). The AjapORco was grouped into extremely high conservation ORco receptors. Various AjapORs were more closely related to NvitORs and TpreORs than they were to AmelORs ( Figure 1).

Identification of Putative Odorant Receptors
We identified 184 putative OR genes in A. japonicus. The sequence analysis revealed that 92 of 184 sequences were full-length putative OR genes with ORFs with five to eight predicted TMDs and an average length of 1163 bp. The OR co-receptor, named AjapOrco, was also found. Other putative ORs (AjapOR1-AjapOR183) were given names followed by a numeral in descending order of the length of their coding regions. Sequence information for the putative ORs in A. japonicus is listed in Table S2 and File S1.
To further assess the relationships between the A. japonicus ORs and the known Hymenoptera ORs, we carried out an ML tree analysis using the putative AjapORs and ORs of T. pretiosum, N. vitripennis, and A. mellifera ( Figure 1). The AjapORco was grouped into extremely high conservation ORco receptors. Various AjapORs were more closely related to NvitORs and TpreORs than they were to AmelORs ( Figure 1).

Identification of Putative Ionotropic Receptors
A total of 17 putative IRs were obtained by searching the transcriptome of A. japonicus and annotated by BLASTx. Of these IRs, 11 sequences contained full-length ORFs, from 399 to 1004 amino acids. The remaining six sequences were incomplete due to the lack of

Identification of Putative Ionotropic Receptors
A total of 17 putative IRs were obtained by searching the transcriptome of A. japonicus and annotated by BLASTx. Of these IRs, 11 sequences contained full-length ORFs, from 399 to 1004 amino acids. The remaining six sequences were incomplete due to the lack of a 5 and/or 3 terminus. A total of 15 IRs contained more than three TMDs as predicted by TMHMM 2.0, which was consistent with the characteristics of insect IRs. All 15 AjapIRs were named based on their orthologous relationships with the IRs from D. melanogaster, except for AjapIR75f.1 and AjapIR 75f.2, named based on their homology with A. mellifera. The sequence information of the putative IRs in A. japonicus is listed in Table S2 and File S1.
To further assess the relationships between the A. japonicus IRs and known Hymenoptera IRs, we carried out an ML tree analysis using the putative AjapIRs and IRs of N. vitripennis, A. mellifera, and D. melanogaster (Figure 2). In the IRs ML tree, obviously, the co-receptors AjapIR8a and AjapIR25a clustered to form the IR8a and IR25a evolutionary branches, respectively. The IR8a/25a subset clustered with the N-methyl-D-aspartate (NMDA) and nonNMDA iGluRs subset. The remaining IRs clustered on different branches (i.e., IR64a, 75, and 93a) of other species. Significant separation of NMDA (N-methyl-Daspartate) and non-NMDA iGluRs from IRs was found. Additionally, we found impressive IR duplications in the co-receptor IR25a (2 sequences), IR64a (2 sequences), and IR75 (4 sequences) subfamilies ( Figure 2). a 5′ and/or 3′ terminus. A total of 15 IRs contained more than three TMDs as predicted by TMHMM 2.0, which was consistent with the characteristics of insect IRs. All 15 AjapIRs were named based on their orthologous relationships with the IRs from D. melanogaster, except for AjapIR75f.1 and AjapIR 75f.2, named based on their homology with A. mellifera. The sequence information of the putative IRs in A. japonicus is listed in Table S2 and File S1.
To further assess the relationships between the A. japonicus IRs and known Hymenoptera IRs, we carried out an ML tree analysis using the putative AjapIRs and IRs of N. vitripennis, A. mellifera, and D. melanogaster (Figure 2). In the IRs ML tree, obviously, the co-receptors AjapIR8a and AjapIR25a clustered to form the IR8a and IR25a evolutionary branches, respectively. The IR8a/25a subset clustered with the N-methyl-D-aspartate (NMDA) and nonNMDA iGluRs subset. The remaining IRs clustered on different branches (i.e., IR64a, 75, and 93a) of other species. Significant separation of NMDA (Nmethyl-D-aspartate) and non-NMDA iGluRs from IRs was found. Additionally, we found impressive IR duplications in the co-receptor IR25a (2 sequences), IR64a (2 sequences), and IR75 (4 sequences) subfamilies ( Figure 2).

Transcription Profiling of the Olfactory Receptor Genes
The expression profiles of 184 ORs were examined in different tissues of A. japonicus based on the TPM values. All the OR genes were exclusively expressed in the antennae of both sexes, and almost no expression (TPM value less than 1) in the female abdomen was observed, except for AjapOR77, which was 8.92. The TPM values for these genes ranged from 0 to 206.82 in the female antennae and from 0 to 393.93 in the male antennae ( Figure 3). Of these ORs, 165 ORs, except AjapOR77, had upregulated expression in the antennae ( Figure 4A). Among those expressed in both female and male antennae, 104 ORs were predominantly expressed in the female, whereas 29 ORs including AjapORco (FAn: 28.37, MAn: 154.62) were predominantly expressed in the male. The remaining 51 ORs were roughly equally expressed in both female and male antennae ( Figure 4B).

Transcription Profiling of the Olfactory Receptor Genes
The expression profiles of 184 ORs were examined in different tissues of A. japonicus based on the TPM values. All the OR genes were exclusively expressed in the antennae of both sexes, and almost no expression (TPM value less than 1) in the female abdomen was observed, except for AjapOR77, which was 8.92. The TPM values for these genes ranged from 0 to 206.82 in the female antennae and from 0 to 393.93 in the male antennae ( Figure  3). Of these ORs, 165 ORs, except AjapOR77, had upregulated expression in the antennae ( Figure 4A). Among those expressed in both female and male antennae, 104 ORs were predominantly expressed in the female, whereas 29 ORs including AjapORco (FAn: 28.37, MAn: 154.62) were predominantly expressed in the male. The remaining 51 ORs were roughly equally expressed in both female and male antennae ( Figure 4B).  Similarly, the expression profiles of the 17 IRs were examined ( Figure 5). The TPM values for these genes ranged from 0.04 to 1.84 in the female abdomen (except for AjapGlu-R1B and AjapGlu-R1, which were 2.63 and 3.42, respectively), from 0.06 to 124.45 in the female antennae, and from 0.49 to 281.66 in the male antennae ( Figure 5A). Among those expressed in the female, 15 IRs genes had upregulated expression in the antennae. The AjapGlu-R1B and AjapGlu-R1 were upregulated in the abdomen ( Figure 5B). Among those expressed in both the female and male antennae, AjapIR25a.2 and AjapIR93a were upregulated in the female compared to the male, while eight IRs had upregulated expression in the male compared to the female ( Figure 5C). Plants 2023, 11, x FOR PEER REVIEW 8 of 13 Similarly, the expression profiles of the 17 IRs were examined ( Figure 5). The TPM values for these genes ranged from 0.04 to 1.84 in the female abdomen (except for AjapGlu-R1B and AjapGlu-R1, which were 2.63 and 3.42, respectively), from 0.06 to 124.45 in the female antennae, and from 0.49 to 281.66 in the male antennae ( Figure 5A). Among those expressed in the female, 15 IRs genes had upregulated expression in the antennae. The AjapGlu-R1B and AjapGlu-R1 were upregulated in the abdomen ( Figure 5B). Among those expressed in both the female and male antennae, AjapIR25a.2 and AjapIR93a were upregulated in the female compared to the male, while eight IRs had upregulated expression in the male compared to the female ( Figure 5C).   Similarly, the expression profiles of the 17 IRs were examined ( Figure 5). The TPM values for these genes ranged from 0.04 to 1.84 in the female abdomen (except for AjapGlu-R1B and AjapGlu-R1, which were 2.63 and 3.42, respectively), from 0.06 to 124.45 in the female antennae, and from 0.49 to 281.66 in the male antennae ( Figure 5A). Among those expressed in the female, 15 IRs genes had upregulated expression in the antennae. The AjapGlu-R1B and AjapGlu-R1 were upregulated in the abdomen ( Figure 5B). Among those expressed in both the female and male antennae, AjapIR25a.2 and AjapIR93a were upregulated in the female compared to the male, while eight IRs had upregulated expression in the male compared to the female ( Figure 5C).

Discussion
Although A. japonicus is an important biological control factor for fruit crop pests, with success in the biocontrol of Pentatomidae pests such as H. halys [5], the chemical ecology of this group and the molecular basis of its olfaction are still largely unknown. In this study, we reported the sequencing, assembly, and annotation of the antennal and abdominal transcriptomes in A. japonicus, and we identified 201 olfactory receptor genes. We also assayed the tissue-specific and sex-biased expression profiles of those genes based on transcriptome profiling using RNA sequencing (RNA-seq) data. The data provide valuable information for further studies on the molecular mechanisms of A. japonicus chemoperception.
We identified 184 ORs in the antennal and abdominal transcriptomes, which was more than previously reported for Sirex noctilio Fabricius (Hymenoptera: Symphyta) (41 ORs) [52], T. japonicum (51 ORs) [18], T. pretiosum (105 ORs) [46], and Cotesia vestalis (Braconidae) (Hymenoptera: Braconidae) (158 ORs) [53], and fewer than reported for Campoletis chlorideae Uchida (Hymenoptera: Ichneumonidae) (211 ORs) [15], Aenasius bambawalei Hayat (Hymenoptera: Encyrtidae) (226 ORs) [54], and N. vitripennis (301 ORs) [47]. The differences in the numbers of identified OR genes could be attributed to the differences in sequencing methods and depth or sample preparation between this and the other studies [55]. Similar limitations also applied in the identification of IRs from A. japonicus in this study. In addition, studies have shown that the number of OR genes have an association with the range width of the host [56,57]. Presumably, a large number of ORs can enhance A. mellifera olfactory abilities, including perception of several pheromone blends, kin recognition signals, and diverse floral odors [49]. This suggests that the remarkably large repertoire of Ors in A. japonicus presumably underlies their remarkable olfactory abilities, including mate and host recognition. The results of the phylogenetic analysis showed that AjapORs were more closely related to NvitORs and TpreORs than they were to AmelORs, which was consistent with the evolutionary relationship between wasps and bees [58,59]. Although the sequences of the insect ORs are highly diverse, we found various ORs from A. japonicus and N. vitripennis that had over 50.00% sequence similarities; in particular, AjapOR11/NvitOR2 and AjapOR32/NvitOR10 shared 76.72% and 75.37% sequence similarity, respectively, suggesting that they have some common and possibly identical olfactory functions. N. vitripennis is the most widely studied of the parasitoid wasps, and the remarkably large repertoire of Ors in N. vitripennis suggests that its chemical ecology is extremely complicated [47]. Presumably, the chemical ecology in A. japonicus is more complicated than currently understood, much as was concluded for N. vitripennis. In addition, AjapORco/NvitORco shared 89.26% sequence similarity, which was consistent with the fact that the amino acid sequence of ORco is highly conserved among different species [57,60].
The tissue-expressed and sex-expressed profiles of ORs correspond to their biological functions and increase our understanding of the parasitoid olfaction system at the molecular level [15,41,[61][62][63]. Of the ORs, 165 ORs, except AjapOR77, were upregulated in the female antennae compared to the female abdomen, which may be involved in the antennal recognition processes for host-searching, mating, and other behaviors in A. japonicus. Most noteworthy, AjapOR77 was highly expressed in both the antennae and the abdomen of female, which is similar to previous reports that OR genes were expressed in some insect non-olfactory tissues, such as the ovipositor [41,64,65]. The HassOR31 had high expression in the ovipositor of Helicoverpa assulta (Guenée) (Lepidoptera: Noctuidae) and was tuned to Z-3-hexenyl butyrate, which helps females to determine precise egg-laying sites in host plants [41]. This suggests that AjapOR77 may play an important role in the recognition of host odorant molecules or suitable sites for oviposition. In addition, 104 ORs were predominantly expressed in the female antennae compared to the male antennae, suggesting that they may be involved in host habitat location, host location, and host acceptance. Further, the specific functions remain to be further explored. Multiple approaches have been developed for functional characterization of ORs [41].In vitro (e.g., heterologous expression in Xenopus oocytes with two-electrode voltage clamp system, transgenic Drosophila with single sensillum recording (SSR) technique, or cell line expression systems with calcium imaging) and in vivo (e.g., RNA interference (RNAi) or clustered regularly interspaced short palindromic repeats (CRISPR)) functional characterization will help demonstrate their roles in the A. japonicus olfactory system.
Compared with the ORs, the IRs are another type of olfactory receptor in the ORNs [31,32,53]. IRs were first reported as a novel family of insect olfactory receptors in D. melanogaster [21,31,32]. We identified 17 IRs in the antennal and abdominal transcriptomes. Similar to Orco, IR8a, IR25a, and IR93a are predicted to act as co-receptors in the IR group because they were co-expressed along with other IRs [21,66,67]. Interestingly, IR duplications in the co-receptor IR25a (AjapIR25a.1 and AjapIR25a.2) were discovered. A similar expression pattern was also reported in N. vitripennis [47]. The functions of these genes, which have been mainly studied in D. melanogaster, include sensing odor, taste, temperature, humidity, and salt [23,33,34,68]. Most IRs tend to be highly expressed in the antennae, and AjapIR25a.2 and AjapIR93a were more expressed in the females than in the males. This indicated that AjapIR25a.2 and AjapIR93a may play important roles in host location or male pheromone sensation. Both temperature and humidity perception in Drosophila are dependent on IR93a and IR25a, and these two receptors both play important roles in temperature and humidity perception. For example, IR93a, IR21a, and IR25a were co-expressed in Drosophila larvae, which could feel cold environments. IR93a, IR25a, and IR40a are co-expressed in Drosophila and can sense humidity in the environment [68]. Therefore, it can be speculated that AjapIR93a and AjapIR25a also have a similar function, that is, they can sense both temperature and humidity, and thus regulate a series of A. japonicus behaviors. In D. melanogaster, male-biased patterns of IR expression as well as functional analyses revealed that both IR52c and IR52d may determine male copulation [31]. Therefore, it is presumed that the eight AjapIRs (IR8a, 25a.1, 64a.1, 64a.2, 75d.1, 75d.2, 75f.1, and 84a) that showed male-biased expression could play a role in sexual behavior. The specific functions remain to be further explored.

Conclusions
In summary, we sequenced and annotated the olfactory receptor genes in the antennal and abdominal transcriptomes in A. japonicus. A total of 201 olfactory receptor genes including 184 ORs and 17 IRs were identified in the antennal and abdominal transcriptomes. We also assayed the tissue-specific and sex-biased expression profiles of those genes based on the transcriptional level. In total, 165 ORs and 15 IRs were upregulated in the antennae. The expression levels of 133 ORs including AjapORco and 10 IRs including AjapIR8a were significantly different between the female and male antennae. This suggests a range of diverse functions of insect antennae, which, to a greater degree, may facilitate the survival of insects in environments full of chemicals from hosts, host habitat, and mates. The data from the present study may also provide valuable information for further studies on the molecular mechanisms of A. japonicus chemoperception, to ultimately improve pest control measures by using natural enemies, which can be important in an IPM strategy.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12040915/s1, Table S1. Summary of the functional annotations of Anastatus japonicus coding genes. Table S2. Sequence information of the putative odorant receptors in Anastatus japonicus. Table S3. Sequence information of the putative ionotropic receptors in Anastatus japonicus. File S1. The amino acid sequences of the odorant receptors and ionotropic receptors identified in this study.

Conflicts of Interest:
The authors declare no conflict of interest.